DNA sequencing techniques require denaturing polyacrylamide gels. The gels are typically formed between two closely spaced flat glass plates. The bottom and edges of the plates are sealed as best they can be and the gel is then poured into or injected in the space between the plates. The gel then polymerizes in situ.
Standard techniques for preparing the plates are described in Molecular Cloning--A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press (second edition 1989), and Current Protocols in Molecular Biology, Ausubel et al., Greene Publishing Associates and Wyle-Interscience, New York, N.Y., 1991, supplement 16, unit 7.6 (Stalko, B.). The techniques describe processes in which two spacer strips are placed along the sides of the bottom plate, and in some instances a lower spacer is placed along the bottom edge of the lower plate. The top plate is then carefully placed on top of the lower plate. The corners where the spacers meet are then typically sealed with agarose or vacuum grease. After the top plate is placed on the bottom plate the edges are bound with one or more layers of electrical tape in an attempt to make a water tight seal and binder clips are placed on the sides and bottom.
These procedures, however, suffer from a number of drawbacks that make them difficult at best. First, a lot of technician's time is spent in aligning the two or three spacers between the glass plates, and attempting to seal the joints between the spacers with grease or applying tape without wrinkles to the sides and bottoms of the plates so as to make a tight seal. Even after all this time and care, the gel solution frequently leaks out from the bottom corners even for experienced technicians. Since the solution is expensive, messy and neurotoxic, extra time and care is then required in clean up.
Additionally, these techniques require that the flat side of a shark's tooth comb, or a comb with preformed wells, be inserted between the plates at the top edge after the gel solution is poured but before it polymerizes in order to form a flat surface at the top of the gel or to form sample introduction wells n the gel, respectively. This step also requires operator expertise. Great care must be taken that the comb is inserted far enough, but not too far and that bubbles of air are not trapped between the comb and the gel. Furthermore, inserting the comb causes some of the solution to spill out the top of the plates, adding to the clean up effort.
After the gel polymerizes, the extraneous polyacrylamide must be removed from around the combs. If a bottom spacer is used, it must be removed, resulting in an air space between the plates at the bottom of the gel. Since there must be a conductive path through the gel into the buffer solution in which the plates are placed, when the plates are lowered into the buffer solution this air space must be carefully filled with buffer. This is usually accomplished by squirting buffer between the plates using a syringe with a bent needle, which takes additional time and expertise. In addition, if all of the air bubble is not carefully removed, a portion of the gel will not be properly subjected to the electrophoresis voltage, thereby ruining a portion of the results, creating additional waste and cost.